| Rice Black-streaked Dwarf Virus Preparation and Infection on Rice
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Author:
Date:
2017-12-20
[Abstract] Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, making it difficult to construct infectious clones for functional studies in plants. Here we describe a method for inoculating and maintaining RBSDV on rice in a greenhouse for use in laboratory research. The protocol uses SBPHs mass reared in the laboratory. We also describe in detail the propagation of a healthy planthopper population, the preparation of plant material, RBSDV inoculation and the evaluation of the rice after inoculation.
[摘要] 水稻黑条矮缩病毒（RBSDV）属于呼肠孤病毒科的Fijivirus属的成员，感染水稻，玉米，大麦和小麦，可严重影响作物产量。 RBSDV以持久的方式由小型褐飞虱（ Ladellphax striatellus ，SBPH）传播。 RBSDV具有10个线性dsRNA基因组区段，使得构建用于植物功能研究的感染性克隆变得困难。 在这里我们介绍一种在温室中用于在实验室研究中使用的RBSDV接种和维持方法。 该协议使用在实验室饲养的大量的SBPHs。 我们还详细描述了一个健康的稻飞虱种群的繁殖，植物材料的制备，RBSDV接种和接种后水稻的评价。
【背景】水稻黑条矮缩病毒（RBSDV）是Fijivirus属家族中的第二个家族中的公认成员，严重影响东亚水稻和玉米的生产。 （Shikata和Kitagawa，1977; Zhang等人，2001a）。 RBSDV以持续的方式（Shikata和Kitagawa，1977）被小型褐飞虱（灰飞虱，SBPH）传播传播到水稻，玉米，大麦和小麦。 SBPH可从新鲜或冷冻感染的水稻叶片获得RBSDV并将其传递至健康植物（Shikata和Kitagawa，1977; Li等人，2010）。一旦SBPH已经获得病毒，病毒可以在昆虫载体中繁殖，并且可以在昆虫的整个生命周期中传播给宿主植物。然而，SBPH不能透传病毒（Boccardo and ...
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| RNA Chromatin Immunoprecipitation (RNA-ChIP) in Caenorhabditis elegans
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Author:
Date:
2014-12-20
[Abstract] The RNA chromatin immunoprecipitation assay (RNA-ChIP) allows detection and quantification of RNA–protein interactions using in vivo cross-linking with formaldehyde followed by immunoprecipitation of the RNA–protein complexes. Here we describe the RNA–ChIP protocol that we have adapted for Caenorhabditis elegans (C. elegans) to detect interaction between the nuclear Argonaute CSR-1 (chromosome segregation and RNAi deficient) protein and its target nascent RNAs. We have used a transgenic strain expressing a recombinant long isoform of CSR-1 protein fused with N-terminal 3x FLAG epitope.
[摘要] RNA染色质免疫沉淀测定（RNA-ChIP）允许使用体内与甲醛交联，随后RNA-蛋白复合物的免疫沉淀来检测和定量RNA-蛋白质相互作用。 在这里我们描述我们已经适应于秀丽隐杆线虫（ C. elegans ）的RNA-ChIP协议以检测核Argonaute CSR-1（染色体分离和RNAi缺陷 ）蛋白及其目标新生RNA。 我们使用表达与N-末端3x FLAG表位融合的CSR-1蛋白的重组长同种型的转基因菌株。
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| In vitro Transcription (IVT) and tRNA Binding Assay
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Author:
Date:
2014-09-20
[Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the “natural” situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The ...
[摘要] 该方案描述了（i）"活"体外RNA转录与（ii）通过放射性标记的预先形成的tRNA的结合，然后是天然凝胶电泳和磷光成像仪扫描以显现复合物的偶联。 必要性来自一种RNA在不存在其相互作用配偶体时形成的稳定结构。 T盒前导RNA，转录控制系统，折叠成热力学非常稳定的茎 - 环结构，没有tRNA存在，这使得体外结合两个预先形成的RNA的相互作用非常困难。 因此，我调整结合测定以模拟细菌细胞中的"天然"情况，其中预先形成的稳定的tRNA已经存在，而T盒前导RNA被RNA聚合酶主动转录。 方案的第一部分还描述了体外转录和tRNA的标记。
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